CN115368266B - 一种丹参酚酸a衍生的生物活性探针及其制备方法和应用 - Google Patents
一种丹参酚酸a衍生的生物活性探针及其制备方法和应用 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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Abstract
本发明提供了一种丹参酚酸A衍生的生物活性探针及其制备方法和应用,该探针以丹参酚酸A和丙炔胺为原料,在HATU的催化下,通过将丹参酚酸A的羧基与丙炔胺的氨基发生缩合反应,合成丹参酚酸A探针A‑SA。细胞水平的抗炎和抗氧化实验显示该丹参酚酸A衍生的生物探针在抗炎和抗氧化活性上与丹参酚酸A原型并无显著性差异,且该探针联合荧光标记染料可用于丹参酚酸A在细胞内或动物组织内分布的显示,因此该探针可以用于鉴定丹参酚酸A的体内分布及相关药效学评价研究。
Description
技术领域
本发明属于化学生物学领域,尤其是涉及一种丹参酚酸A衍生的生物活性探针及其制备方法和应用。
背景技术
丹参酚酸A是中药材丹参中重要的活性化合物,用于治疗心绞痛、冠心病、胸闷等疾病,香丹注射液、丹参注射液、丹红注射液等中成药里面均含有丹参酚酸A。丹参酚酸A具有众多药效活性如抗氧化,抗炎,抗细胞凋亡,改善心、脑的缺血再灌损伤,血管内皮保护等作用。丹参酚酸A经静脉注射后,主要以原型发挥作用,后经甲基化代谢和葡萄糖醛酸代谢后排出体外。
然而,丹参酚酸A在细胞和组织内的分布情况未知,其靶点蛋白尚不明确,这阻碍了丹参酚酸A药理机制的深入阐明和药效的全面评价,也是基于丹参酚酸A的新药研发过程中的瓶颈问题。对于丹参酚酸A药理机制的研究还集中在活性指标评价方面,对丹参酚酸A在细胞和组织内分布及靶点蛋白鉴定工作未见报道,这是由于缺乏有效的生物活性探针,这种探针既要保留丹参酚酸A活性母核结构确保其生物活性不受影响;又要引入有效的化学基团,使得丹参酚酸A在细胞和组织内可视化,并确保靶点蛋白能够从复杂的细胞裂解液体系中分离出来。总之,开发兼顾丹参酚酸A药效活性,使其在细胞或组织内可视,并有分离能力的丹参酚酸A生物活性探针(A-SA),是本领域技术人员急需解决的问题。
发明内容
有鉴于此,本发明目的是开发一种能在细胞和组织内可视,并能够用于靶点蛋白鉴定的丹参酚酸A生物活性探针,设计并公开了以丹参酚酸A为原料制备丹参酚酸A生物活性探针的方法及应用。
为达到上述目的,本发明的技术方案是这样实现的:
一种丹参酚酸A衍生的生物活性探针,所述探针为黄色粉末,分子式为C29H25NO9,分子量为531.1529,结构式如下:
优选地,所述探针在5×10-4mol/L以下浓度范围内对细胞活力无影响。
优选地,所述探针在10-5~10-8mol/L浓度范围内具有良好的抗炎活性。
优选地,所述探针在10-5~10-7mol/L浓度范围内具有良好的抗氧化活性。
本发明的第二目的在于提供一种丹参酚酸A衍生的生物活性探针的制备方法,以丹参酚酸A和丙炔胺为原料,经HATU的催化,在丹参酚酸A的9号位通过缩合反应引入炔基侧链,从而合成丹参酚酸A生物活性探针。
所述的探针用于显示丹参酚酸A在细胞和/或动物组织中的分布,从而可以在细胞和动物水平示踪丹参酚酸A。
所述的探针用于捕获丹参酚酸A的靶点蛋白。
相对于现有技术,本发明的优点和有益效果:
本发明所制备的丹参酚酸A生物活性探针(A-SA),具有良好的抗炎、抗氧化活性,可用于观察丹参酚酸A在细胞和组织内的分布,可用于丹参酚酸A靶点蛋白的鉴定和药效机制的阐释,为相关药效学评价提供新的研究策略和方法,具有良好的应用前景。用本发明合成丹参酚酸A生物活性探针的方法,原料易得,合成成本很低,操作方法简单,条件较为温和,适用于工业放大。
附图说明
构成本发明的一部分的附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。在附图中:
图1为丹参酚酸A生物活性探针(A-SA)的结构图;
图2为丹参酚酸A生物活性探针(A-SA)的合成路线;
图3为丹参酚酸A生物活性探针(A-SA)的核磁共振结果,其中A为核磁共振氢谱图;B为核磁共振碳谱图;
图4为丹参酚酸A生物活性探针(A-SA)的高分辨质谱结果;
图5为丹参酚酸A生物活性探针(A-SA)在H9C2细胞上的细胞毒性考察;#p<0.05,与对照组相比较;
图6为丹参酚酸A生物活性探针(A-SA)的抗炎活性分析,其中A为双荧光基因报告分析实验结果,NF-κB抑制率(%);B为Western blot结果,###p<0.001,与空白对照组相比较;***p<0.001,与模型组相比较;
图7为丹参酚酸A生物活性探针(A-SA)的抗氧化活性分析,其中A为双荧光基因报告分析实验结果,NRF2诱导率;B为Western blot结果,***p<0.001,*p<0.05,与对照组相比较;
图8为丹参酚酸A生物活性探针(A-SA)在心肌H9C2细胞上分布情况;
图9为丹参酚酸A生物活性探针(A-SA)在心肌组织上分布情况;
图10为丹参酚酸A实现荧光显色的化学反应原理;
图11为丹参酚酸A生物活性探针(A-SA)靶点蛋白捕获的化学反应原理;
图12为丹参酚酸A生物活性探针(A-SA)在心肌H9C2细胞上的蛋白捕获研究结果。
具体实施方式
除有定义外,以下实施例中所用的技术术语具有与本发明所属领域技术人员普遍理解的相同含义。以下实施例中所用的试验试剂,如无特殊说明,均为常规生化试剂;所述实验方法,如无特殊说明,均为常规方法。
下面结合实施例及附图来详细说明本发明。
实施例1:丹参酚酸A生物活性探针(A-SA)的制备方法
实验方法如下
准确称量丹酚酸(494mg,1mmoL)溶于N,N-二甲基甲酰胺(DMF)中,加入HUTU(760mg,2mmoL),室温搅拌30min,缓慢加入丙炔胺(110mg,2mmoL),室温搅拌过夜,TLC检测原料D:M=10:1。加入1mol/L稀盐酸猝灭反应,使用乙酸乙酯与饱和食盐水萃取三次,合并有机相,旋干,干发上样,使用100-200目硅胶,得到黄色固体355mg,产率72%(合成化学式如图2)。
如图3所示,终产物丹参酚酸A生物活性探针(A-SA)的核磁共振检测(NMR)检测结果为:1H NMR(400MHz,DMSO-d6)δ9.98(s,1H),9.05(d,J=37.4Hz,2H),8.72(d,J=21.6Hz,3H),8.57(t,J=5.6Hz,1H),7.88(d,J=15.8Hz,1H),7.18(d,J=8.5Hz,1H),7.07–6.97(m,2H),6.83–6.71(m,3H),6.64(d,J=2.1Hz,1H),6.59(d,J=8.0Hz,1H),6.51(d,J=16.3Hz,1H),6.45(dd,J=8.1,2.1Hz,1H),6.31(d,J=15.8Hz,1H),5.07(dd,J=9.0,4.0Hz,1H),3.87(dd,J=5.9,2.5Hz,2H),3.11(t,J=2.5Hz,1H),2.89(dd,J=14.1,4.0Hz,1H),2.80–2.73(m,1H).13C NMR(101MHz,DMSO-d6)δ168.92,165.99,147.16,145.77,145.49,144.94,144.63,143.92,143.15,135.76,128.94,127.48,127.01,123.64,120.14,119.14,118.92,118.80,116.56,115.76,115.33,114.50,114.16,112.85,80.87,74.00,73.12,36.97,27.92。
对丹参酚酸A生物活性探针(A-SA)进行高分辨质谱(HRMS)检测,[M+Na+]+的高分辨质谱HRMS计算值为:554.1422,实测值:554.1409(图4)。
结合NMR和HRMS鉴定结果,该化合物为目标产物。该丹参酚酸A生物活性探针(A-SA)为黄色粉末,分子式为C29H25NO9,分子量为531.1529,易溶于水。
实施例2:丹参酚酸A生物活性探针(A-SA)的细胞毒性考察
实验方法如下
使用Cell Counting Kit-8(CCK-8)试剂盒,检测不同剂量A-SA对细胞活性的影响,实验方法如下:
1)将复苏后的H9C2细胞置于DMEM高糖培养基中(含10%FBS和1%双抗),置于5%CO2培养箱中,37℃恒温培养。待细胞生长至融合度为50~60%时,每孔加入100mL含不同浓度丹参素生物活性探针的无血清培养基,孵育过夜;弃去培养基,加入10μL的CCK-8溶液和90μL PBS溶液的混合液;将培养板置于培养箱内孵育30分钟;按照试剂盒说明书,用酶标仪(Spark,奥地利)测定其吸光度,检测波长为450nm。
2)实验设置不含药物的对照组,以及不含细胞和药物的空白组进行校准。实验重复6次,取平均值,通过方差分析统计探针对细胞活力的影响。计算公式:细胞存活率=[(As-Ab)/(Ac-Ab)]×100%。其中,As表示实验组吸光度;Ac表示对照组吸光度;Ab表示空白组吸光度。
3)实验结果如图5所示,在A-SA浓度小于5×10-4mol/L时,A-SA对H9C2心肌细胞的毒性较小,当浓度超过5×10-4mol/L时H9C2心肌细胞的存活率有所下降。因此,选择5×10- 4mol/L以下浓度A-SA为细胞水平实验的安全剂量。
实施例3:丹参酚酸A生物活性探针(A-SA)的抗炎活性考察
实验方法如下
1)配LB培养基(胰蛋白胨10g/L,酵母提取物5g/L,氯化钠10g/L)200mL,用NaOH调节pH使其达到7.4。加5mL LB培养基到试管中,置于灭菌锅中灭菌后加20uL含质粒的菌体(PGL4.32和Renilla)和10uL氨苄,放置于摇床12h后,使用质粒小提试剂盒提取质粒PGL4.32和Renilla。
2)将HEK293T细胞培养于96孔细胞培养板中,待细胞汇合度在50-70%时进行转染。将转染试剂PEI(1mg/mL)、内参荧光素酶报告基因质粒Renilla(9.6ng/孔)和荧光素酶报告基因质粒pGL4.32(100ng/孔)按比例混匀,其中PEI:pGL4.32=6:1(质量比),静置15min使之充分反应。吸弃旧培养基,加入静置后的混合液(100μL/孔)共转染细胞。质粒转染24h后进行加药处理。
将TNF-α用灭菌水稀释至2000ng/mL的母液,Dex(Dexamethasone,低塞米松)稀释至10-1mol/L的母液。实验分为空白对照组(control,DMEM高糖培养基),模型组(model,DMEM高糖培基稀释终浓度为20ng/mL的TNF-α),阳性对照组(Dex,DMEM高糖培基稀释终浓度为10-5mol/L的Dex+终浓度为20ng/mL的TNF-α),丹参酚酸A生物活性探针(A-SA)剂量组(DMEM高糖培基稀释终浓度为10-5、10-6、10-7、10-8mol/L A-SA+终浓度为20ng/mL的TNF-α),各组细胞反应在药物孵育6h后检测。
3)弃去培养液,用磷酸盐缓冲液(PBS,100μL/孔)清洗两次,再加入细胞裂解液(Passive Lysis Buffer,20μL/孔,母液稀释5倍)室温振摇30min。从预处理振摇后的细胞培养板每孔吸出15μL细胞裂解液于1.5mL EP管中,加入20μL预先配置的LAR反应液,混匀后于ModulusTM单管型生物与化学发光、荧光吸收多功能检测仪上检测萤火虫荧光素酶报告基因活性,然后再加入20μL终止液(现配现用),混匀后检测海肾荧光素酶报告基因活性。第一次所测得的萤火虫萤光素酶活性值与第二次所测得的内参萤光素酶活性值的比值,则为校正转染效率后的相对活性值F。抗炎活性(nf-kb的抑制率)=(Fm-Fs)*100%/Fm;Fm是模型组的荧光值,Fs是实验组的荧光值。
4)取对数生长期MAEC,弃去培养基,PBS冲洗3次,加入0.25%胰蛋白酶消化细胞,用含胎牛血清的完全培养基终止消化,充分吹打细胞至均匀的细胞悬液,室温2000rpm离心后,弃去上清液,将细胞用新鲜的完全培养基吹打至均匀的细胞悬液,将细胞悬液分配在6孔板中,于细胞培养箱中待细胞贴壁生长。待细胞密度达到80%左右,向每孔中分别加入样品溶液,使其终浓度为:10-6mol/L的丹参酚酸A,孵育细胞6h,再向每孔的培养基中加入TNF-α使每孔终浓度为20ng/mL,刺激细胞2h。
细胞蛋白样品制备
①配制细胞蛋白裂解液:每1mL高效RIPA裂解液中加入10μL PMSF液和10μL蛋白磷酸酶抑制剂,混匀后在4℃暂存,现配现用。
②将细胞培养液吸弃,后用PBS清洗3次6孔板,每孔加入300μL的蛋白裂解液,置于冰上裂解细胞30min。
③用洁净的细胞刮将细胞和裂解液刮净至孔的一侧,再用移液枪将细胞裂解液全部转移至EP管中,4℃下12000rpm离心10min。离心后取上清夜即为细胞蛋白提取液。将蛋白上清液进行蛋白定量和western blot实验,一抗孵育抗体为抗nf-kb抗体。
5)实验结果如图6所示,丹参酚酸A生物活性探针(A-SA)在10-5~10-8mol/L浓度范围内体现了浓度依赖性的抗炎活性,且A-SA在10-5~10-8mol/L浓度范围内的抗炎活性与丹参酚酸A原型的抗炎活性相比,无显著性差异。
实施例4:丹参酚酸A生物活性探针(A-SA)的抗氧化活性考察实验方法如下
1)配LB培养基(胰蛋白胨10g/L,酵母提取物5g/L,氯化钠10g/L)200mL,用NaOH调节pH使其达到7.4。加5mL LB培养基到试管中,置于灭菌锅中灭菌后加20uL含质粒的菌体(PGL4.37和Renilla)和10uL氨苄,放置于摇床12h后,使用质粒小提试剂盒提取质粒PGL4.37和Renilla。
2)将HEK293T细胞培养于96孔细胞培养板中,待细胞汇合度在50-70%时进行转染。将转染试剂PEI(1mg/mL)、内参荧光素酶报告基因质粒Renilla(9.6ng/孔)和荧光素酶报告基因质粒pGL4.32(100ng/孔)按比例混匀,其中PEI:pGL4.32=6:1(质量比),静置15min使之充分反应。吸弃旧培养基,加入静置后的混合液(100μL/孔)共转染细胞。质粒转染24h后进行加药处理。
实验分为空白对照组(control,DMEM高糖培养基),阳性对照组(tBHQ,DMEM高糖培基稀释终浓度为10-6mol/L的tBHQ),丹参酚酸A生物活性探针(A-SA)剂量组(DMEM高糖培基稀释终浓度为10-5、10-6、10-7mol/L A-SA),各组细胞反应在药物孵育6h后检测。
3)弃去培养液,用磷酸盐缓冲液(PBS,100μL/孔)清洗两次,再加入细胞裂解液(Passive Lysis Buffer,20μL/孔,母液稀释5倍)室温振摇30min。从预处理振摇后的细胞培养板每孔吸出15μL细胞裂解液于1.5mL EP管中,加入20μL预先配置的LAR反应液,混匀后于ModulusTM单管型生物与化学发光、荧光吸收多功能检测仪上检测萤火虫荧光素酶报告基因活性,然后再加入20μL终止液(现配现用),混匀后检测海肾荧光素酶报告基因活性。第一次所测得的萤火虫萤光素酶活性值与第二次所测得的内参萤光素酶活性值的比值,则为校正转染效率后的相对活性值F,表征抗氧化活性(nrf2的表达水平)。
4)取对数生长期MAEC,弃去培养基,PBS冲洗3次,加入0.25%胰蛋白酶消化细胞,用含胎牛血清的完全培养基终止消化,充分吹打细胞至均匀的细胞悬液,室温2000rpm离心后,弃去上清液,将细胞用新鲜的完全培养基吹打至均匀的细胞悬液,将细胞悬液分配在6孔板中,于细胞培养箱中待细胞贴壁生长。待细胞密度达到80%左右,向每孔中分别加入样品溶液,使其终浓度为:10-6mol/L的丹参酚酸A和10-6mol/L的tBHQ,孵育细胞6h。
细胞蛋白样品制备
①配制细胞蛋白裂解液:每1mL高效RIPA裂解液中加入10μL PMSF液和10μL蛋白磷酸酶抑制剂,混匀后在4℃暂存,现配现用。
②将细胞培养液吸弃,后用PBS清洗3次6孔板,每孔加入300μL的蛋白裂解液,置于冰上裂解细胞30min。
③用洁净的细胞刮将细胞和裂解液刮净至孔的一侧,再用移液枪将细胞裂解液全部转移至EP管中,4℃下12000rpm离心10min。离心后取上清夜即为细胞蛋白提取液。将蛋白上清液进行蛋白定量和western blot实验,一抗孵育抗体为抗nrf2抗体。
5)实验结果如图7所示,丹参酚酸A生物活性探针(A-SA)在10-5~10-7mol/L浓度范围内体现了浓度依赖性的抗氧化活性,且A-SA在10-5~10-8mol/L浓度范围内的抗氧化活性与丹参酚酸A原型的抗氧化活性相比,无显著性差异。
实施例5:丹参酚酸A生物活性探针(A-SA)在心肌细胞中的荧光定位考察
实验方法如下
1)将H9C2细胞传代至共聚焦小皿中,细胞生长至融合度约为50%时,将细胞分为2组,分别为:丹参酚酸A组(10μM)、丹参酚酸A生物活性探针(A-SA)组(10μM),分别加入对应的药物后继续培养6h。
2)用PBS洗涤细胞后,加入4%多聚甲醛,室温固定30min,再次用PBS震荡洗涤细胞3次,5min/次。
3)加入10%羊血清,封闭1h,弃去封闭液;随后加入1:1000稀释的一抗溶液,4℃孵育过夜。室温条件下,使用TBST震荡清洗3次,10min/次。
4)室温,TBST清洗3次,30min/次。加入100μMFITC-azide与100μM点击反应催化剂,37℃条件下,避光反应1h。TBST清洗3次,30min/次后,通过激光共聚焦显微镜进行拍摄。化学反应原理如图10所示。
5)实验结果如图8所示,丹参酚酸A主要分布在了H9C2心肌细胞的细胞膜和细胞胞浆中,细胞核中未见丹参酚酸A的分布,这说明丹参酚酸A生物活性探针(A-SA)与FITC-azide联合能显示丹参酚酸A在细胞中的分布。
实施例6:丹参酚酸A生物活性探针(A-SA)在心肌细胞中的荧光定位考察
实验方法如下
1)小鼠分别灌胃给予200mg/kg的丹参酚酸A或200mg/kg的丹参酚酸A生物活性探针(A-SA)。
2)给药4h后断颈处死,剥离心脏,剔除脂肪、结缔组织、血液等杂物,置于盛有PBS的平皿中,清洗后备用。将干净的心脏分别浸泡在4%多聚甲醛中,室温,浸泡24h,固定包埋进行组织切片分析。
3)将组织切片放置在60℃恒温箱中20min,拿出后立即放置在二甲苯中浸泡15min,之后放入新的二甲苯再次浸泡15min,完成脱蜡。将切片依次放置在无水乙醇I和无水乙醇II中各5min,95%、90%、80%和70%乙醇中各2min。随后使用去离子水洗2次,每次5min。将3% H2O2溶液滴加到组织切片上,室温,避光放置10min,然后用去离子水清洗3次,每次5min。
4)将切片放置于柠檬酸盐缓冲液中,采用微波炉加热至溶液沸腾后于95℃条件下保温10min。待溶液冷却至室温后取出切片,使用PBS清洗3次,每次5min,后置于平皿中。
5)加入100μMFITC-azide标签与100μM点击反应催化剂,37℃,避光反应1h,化学反应原理如图10所示。弃去点击反应液,分别用70%甲醇与TBST清洗3次,每次30min。切片进行脱水,依次置于70%、80%、90%、95%和无水乙醇中各2min。最后将切片放置于二甲苯中2次,每次2min。在切片上滴加中性树脂,盖上盖玻片。放置避光处晾干后通过激光共聚焦显微镜进行拍摄。
6)实验结果如图9所示,丹参酚酸A在心肌组织中有明显分布,这说明丹参酚酸A生物活性探针(A-SA)与FITC-azide联合能显示丹参酚酸A在组织中的分布。
实施例7:丹参酚酸A生物活性探针(A-SA)在心肌细胞中的靶点捕获研究
实验方法如下
1)1mL的氨基微型磁球(5mg/mL)溶解在5mL的PBS中,用移液枪吹打几次后用磁铁聚集磁球,将PBS溶液吸弃,如此反复几次,清洗磁球。1mL洗净的氨基微型磁球(5mg/mL)溶解在5mL的PBS中,加入0.5mg Sulfo-SADP混匀,该混合反应液在震荡器上过夜反应12h,获得叠氮修饰的磁球。用磁铁将叠氮修饰的磁球富集后,将反应液吸弃,用水和甲醇交替清洗叠氮修饰磁球各三次,保留叠氮修饰磁球于4℃冰箱。将生长良好的H9C2细胞胰酶消化后,用细胞完全培养液配成细胞悬液接种在6孔板上,待细胞密度生长达80%左右时,分为三组向每组孔中给药分别为:完全培养基,20μmol/L的A-SA,20μmol/L的丹参酚酸A,每个孔重复三次,孵育6h。
2)1mL蛋白裂解液(含PMSF 10μL/mL)在冰上裂解细胞30min,后将细胞蛋白液收集并在4℃,12000rpm离心10min,吸取上清液转移置EP管中,4℃保存待用。将叠氮修饰磁球溶解在600μL蛋白提取液中,并向该溶液中加入CuSO4(终浓度1mmol/L)和维生素C(1mmol/L)混匀后,在振荡器上4℃反应12h。磁铁吸附磁球,将反应液吸弃,PBS清洗磁球3次,将磁球溶解在300μLDL-二硫苏糖醇(DTT)(1mmol/L)中4℃振荡反应30min,磁铁吸附磁球,转移上清液至EP管中,冻干后用20μL蛋白上样缓冲液重新溶解,金属浴上100℃煮5min,总蛋白上样组上样量为20μL蛋白提取液,用来跑10%聚丙烯酰胺凝胶电泳,将聚丙烯酰胺凝胶一块进行银离子染色。本实验的化学反应原理如图11所示。
3)实验结果如图12所示,泳道1中是protein ladder用于显示蛋白分子量。泳道2中是10-5mol/L A-SA与H9C2细胞总蛋白孵育后,从总蛋白中捕获到的蛋白靶点条带。泳道3是H9C2细胞总蛋白对照组。将泳道2与3中的蛋白进行比较,结果显示泳道2中的蛋白条带清晰且较纯净,这说明丹参酚酸A生物活性探针(A-SA)能够实现细胞上蛋白靶点的捕获。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (2)
1.一种丹参酚酸A衍生的生物活性探针,其特征在于:所述探针的分子式为C29H25NO9,分子量为531.1529,结构式如下:
2.权利要求1所述的丹参酚酸A衍生的生物活性探针的制备方法,其特征在于:以丹参酚酸A和丙炔胺为原料,经HATU的催化,在丹参酚酸A的9号位通过缩合反应引入炔基侧链,从而合成丹参酚酸A生物活性探针。
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